Two species (or subunits) of cytochrome P450scc of rat adreal mitochondria are described. Species I has a high affinity for cholesterol and esccbits temperature and pH-dependent spin stage changes. Species 2 has a high affinity for 2OS, 22R-dihydroxycholesterol and does not exhibit temperature or pH-dependent spin stage changes. 20S-and 22R-hydroxycholesterol bind to both species but with distinct spin state changes and binding rates. ACTH activates the binding of cholesterol to species 1. The activationn of corticosterone formation in isolated rat adrenal ce;;s bu ACTH and cAMP was inhibited by five protein synthesis inhibitors to a similar extent to their effect on protein synthesis, and by three inhibitors of microtubule formation. When infsed into hypohysectomized rats with ACTH, the effects on plasma corticosterone and the level of P450scc -cholesterol complexes were exactly parallel. Thus all these inhibitors apppear to decrease cholesterol flux to cytochrome P450. The temporal changes in cytochrome P450scc in the mitochondria of pig corpora lutea during the estrus cycle have been measured and correspond to progesterone output. The heterolytic agent, PGF 2 alpha appears to act initially by decreasing the flux of cholesterol to cytochrome P450scc. BIBLIOGRAPHIC REFERENCES: Jefcoate, Colin R. Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy. J. Biol. Chem. 250:4663-4670 (1975). Jefcoate, Colin R. and Orme-Johnson, William H. Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states. J. Biol. Chem. 250: 4671-4677 (1975).